mNGS assay was carried out as previously described (8). Before viral nucleic acid (NA) isolation, 100 μL of each CSF sample was treated with Turbo DNase (Ambion, Life Technology, ThermoFisher, https://www.thermofisher.com) and RNase I enzyme (Ambion). Then viral NA was isolated using a QIAamp viral RNA kit (QIAGEN GmbH, https://www.qiagen.com), and recovered in 50 μL of elution buffer provided with the extraction kit. Double-stranded DNA was synthesized from the isolated viral NA by using a set of 96 nonribosomal primers (FR26RV–Endoh primers) and then was randomly amplified by using the FR20RV primer (5′-GCCGGAGCTCTGCAGATATC-3′). Finally, the amplified product was subjected to a library preparation step by using Nextera XT sample preparation kit (Illumina, https://www.illumina.com), following the manufacturer’s instructions, and sequenced by using a MiSeq reagent kit, version 3 (600 cycles) (Illumina) in a MiSeq platform (Illumina).

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